1. Field of the Invention
The present invention relates to a compound inhibiting Akt, which is a type of kinase, an agent for inducing apoptosis comprising the compound, and a method of inducing apoptosis. The present invention further relates to a method of screening for a substance inhibiting biological activities such as the expression of pathogenesis factors in microorganisms and the generation of biofilms.
2. Description of Related Art
Apoptosis is the process of physiological cell death that has been proposed by Kerr and Wyllie et al., (see Br. J. Cancer 26, 239-257, 1972 and J. Pathol. 111, 85-94, 1973). Apoptosis is not the simple phenomenon of cell disintegration, but is active cell death programmed by the genes of a cell in order to maintain the life of an individual. Apoptosis plays an important role not only in the formation of a body in the developmental process, but also in normal cell turnover, the maintenance of the nervous system, the establishment of the immune system and the like in a mature individual in order to control the cellular society (see Science 154, 605-612, 1966 and Rev. Cell. Biol. 7, 663-698, 1991). Moreover, it has become clear that apoptosis is involved not only in basic life phenomena, but also closely involved in the onset of various diseases, for example cancer, autoimmune diseases, viral infectious diseases such as AIDS and neurodegenerative diseases such as Alzheimer disease (see Lancet 341, 1251-1254, 1993).
Apoptosis is caused by various apoptosis-inducing factors under physiological and pathological conditions, and is defined by morphological changes and biochemical changes characteristic in apoptotic cells. Apoptosis is distinguished from necrosis, which is a passive disintegration process wherein normal cells that have received extreme injuries, such as from burns or bruises, die.
Factors for apoptosis induction include, for example, biological factors such as signals from hormones or cytokines, and removal of growth factors, as well as physical factors such as radioactive rays and heat, and chemical factors such as drugs. The mechanism varies depending on apoptosis-inducing factors. Finally, through a common process mainly comprising DNA fragmentation, cell death occurs.
Apoptosis is a form of physiological cell death essential for normal development and differentiation, and occurs in individual cells, for example during the cell turnover in normal biological tissue. Accordingly, excessive suppression of apoptosis causes many functional disorders.
Specific examples of such disorders resulting from apoptosis suppression include cancer, proliferative dermatosis, chronic rheumatoid arthritis, HIV infection, hepatitis and renal diseases. There are currently no effective therapeutic agents against these disorders, and agents for treating and improving such conditions, which have a high clinical usefulness, have been desired.
In nature, microorganisms must survive under various environments. They are forced to survive under environments that of course include oligotrophy, high or low temperatures and pH changes. In vivo environment, they are forced to survive under the presence of phagocytes or antimicrobial humoral factors (e.g., complements, antibodies and lysozymes). Under such circumstances, bacteria have acquired mechanisms for sensitively sensing changes in the environment where they exist. It has been reported as one of such mechanisms that microorganisms sense their own concentrations in the environment via specific signalling substances, and cleverly control their various biological activities according to the concentrations. Such a cell-to-cell signalling mechanism is referred to as the quorum sensing system.
In this signalling mechanism, a substance called an autoinducer (AI) is involved. Signalling among microorganisms is conducted via the autoinducer, thereby regulating wide-ranging biochemical and physiological functions such as the promotion of gene transcription activity, the expression of pathogenicity and the production of antibiotics. This quorum sensing has been discovered in many gram-negative bacteria. As a typical autoinducer, acylated homoserine lactone has been reported. Furthermore, acylated homoserine lactone has been revealed to be involved in a wide variety of activities of microorganisms. As these activities, production of exoenzymes in Erwinia carotovora, which is a plant pathogen, and Pseudomonas aeruginosa, which is a causative bacterium of cystic fibrosis, and introduction of Ti plasmid from Agrobacterium tumefaciens to a plant are known.
I-gene, R-gene and target genes, are basically responsible for the quarum sensing system. I-gene encodes an AI synthetic enzyme, and R-gene encodes a transcription activation factor (see Marvin Whiteloy et al., Proc. Natl. Acad. Sci., 96, 13904-13909 (1996)). The autoinducer, that is, a group of substances referred to as acylated homoserine lactone, is synthesized by the AI synthetic enzyme. Acylated homoserine lactone is a molecule that can pass through the outer bacterial membrane. Acylated homoserine lactone is diluted and does not exhibit biological activity when a bacterial concentration in an environment is low. However, as bacterial proliferation proceeds and bacterial density in the environment becomes higher, intra- and extrabacterial concentration of acylated homoserine lactone become higher. When the concentration reaches a certain threshold, binding of acylated homoserine lactone with R-gene product (transcriptional activator) is accelerated. This complex binds to the transcriptional regulatory region of target genes to promote the expression of the target genes, as a result, various biologically active substances are expressed.
It has been reported so far that in clinically important bacteria such as bacteria of the genus Vibrio, Pseudomonas aeruginosa, Serratia and Enterobacter, acylated homoserine lactone promotes the expression of pathogenic factors via the above quorum sensing system. Besides, it has also been shown that acylated homoserine lactone is involved in generation of biofilms by microorganisms (see JP Patent Publication (Kohyo) No. 2002-514092 A).
Biofilms are membranes of organisms, which are generated in an environment with water, particularly on the medial wall surfaces of the duct materials of industrial facilities, the same of domestic piping systems, or the interface on medical transplants, or are generated as lesions of chronic infectious diseases and continue to exist. The biofilm comprises an organic gelatinous structure composed of substrate polymers secreted by resident microorganisms and microorganisms embedded in the structure. The formation of biofilms may limit or completely block the flow in a piping system, or may reduce the lifetime of materials by the corrosive action of embedded bacteria. Control and removal of biofilms from pipe or duct surfaces have been conventionally conducted through the use of corrosive chemical drugs such as chlorine or a strong alkaline solution, or by mechanical means. Such treatments are generally severe for both piping systems and the environment. Microbial resistance against an antimicrobial agent is mainly provided by the protection property of the substrate polymer of a biofilm. In the medical field, the use of high doses of antibiotics has been required for treatment of conditions where biofilms may be involved in. One of the reasons for this is thought to be that the protection ability of biofilm of bacteria is enhanced by extracellular substrate polymers. Therefore, there is a need to control biofilm formation in the medical, environmental and industrial fields.
As described above, the expression of pathogenic factors and biofilm generation are now important issues, so that a method of screening for a substance that inhibits acylated homoserine lactone being involved in these biological activities of microorganisms has been desired. Since acylated homoserine lactone controls various activities in microorganisms, a substance inhibiting acylated homoserine lactone may be screened for by using the survival of microorganisms, or the like as an indicator. However, microorganisms themselves produce acylated homoserine lactone, thus causing bias. This is not preferred as a screening method. Hence, a more effective method of screening for an acylated homoserine lactone-inhibiting substance has been desired.
In the meantime, regarding the effect of an acylated homoserine lactone molecule on animal cells, there have been only a few reports that N-(3-oxododecanoyl)-L-homoserine lactone stimulates IL-8 production in human alveolar cells (see Richard P. Phipps, J. Immunol., 167, 366-374, (2001)) and that the substance promotes TNF-α and IL-12 production in a mouse macrophage culture system (see Gary Telford et al., Infect. Immun., 66, 36-42, (1998)). All of these reports relate to inflammation or immune responses. Both the facts that acylated homoserine lactone inhibits the activities of Akt in animal cells and further induces apoptosis in animal cells have been completely unknown.